Astrocytic Insulin Signaling Couples Brain Glucose Uptake with Nutrient Availability.

We report that astrocytic insulin signaling co-regulates hypothalamic glucose sensing and systemic glucose metabolism. Postnatal ablation of insulin receptors (IRs) in glial fibrillary acidic protein (GFAP)-expressing cells affects hypothalamic astrocyte morphology, mitochondrial function, and circuit connectivity. Accordingly, astrocytic IR ablation reduces glucose-induced activation of hypothalamic pro-opio-melanocortin (POMC) neurons and impairs physiological responses to changes in glucose availability. Hypothalamus-specific knockout of astrocytic IRs, as well as postnatal ablation by targeting glutamate aspartate transporter (GLAST)-expressing cells, replicates such alterations. A normal response to altering directly CNS glucose levels in mice lacking astrocytic IRs indicates a role in glucose transport across the blood-brain barrier (BBB). This was confirmed in vivo in GFAP-IR KO mice by using positron emission tomography and glucose monitoring in cerebral spinal fluid. We conclude that insulin signaling in hypothalamic astrocytes co-controls CNS glucose sensing and systemic glucose metabolism via regulation of glucose uptake across the BBB.

Mechanisms of anion conduction by coupled glutamate transporters.

Excitatory amino acid transporters (EAATs) are essential for terminating glutamatergic synaptic transmission. They are not only coupled glutamate/Na(+)/H(+)/K(+) transporters but also function as anion-selective channels. EAAT anion channels regulate neuronal excitability, and gain-of-function mutations in these proteins result in ataxia and epilepsy. We have combined molecular dynamics simulations with fluorescence spectroscopy of the prokaryotic homolog GltPh and patch-clamp recordings of mammalian EAATs to determine how these transporters conduct anions. Whereas outward- and inward-facing GltPh conformations are nonconductive, lateral movement of the glutamate transport domain from intermediate transporter conformations results in formation of an anion-selective conduction pathway. Fluorescence quenching of inserted tryptophan residues indicated the entry of anions into this pathway, and mutations of homologous pore-forming residues had analogous effects on GltPh simulations and EAAT2/EAAT4 measurements of single-channel currents and anion/cation selectivities. These findings provide a mechanistic framework of how neurotransmitter transporters can operate as anion-selective and ligand-gated ion channels.

Glutamate transporters have a chloride channel with two hydrophobic gates.

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, and its precise control is vital to maintain normal brain function and to prevent excitotoxicity1. The removal of extracellular glutamate is achieved by plasma-membrane-bound transporters, which couple glutamate transport to sodium, potassium and pH gradients using an elevator mechanism2-5. Glutamate transporters also conduct chloride ions by means of a channel-like process that is thermodynamically uncoupled from transport6-8. However, the molecular mechanisms that enable these dual-function transporters to carry out two seemingly contradictory roles are unknown. Here we report the cryo-electron microscopy structure of a glutamate transporter homologue in an open-channel state, which reveals an aqueous cavity that is formed during the glutamate transport cycle. The functional properties of this cavity, combined with molecular dynamics simulations, reveal it to be an aqueous-accessible chloride permeation pathway that is gated by two hydrophobic regions and is conserved across mammalian and archaeal glutamate transporters. Our findings provide insight into the mechanism by which glutamate transporters support their dual function, and add information that will assist in mapping the complete transport cycle shared by the solute carrier 1A transporter family.

Neuronal MML-1/MXL-2 regulates systemic aging via glutamate transporter and cell nonautonomous autophagic and peroxidase activity.

Accumulating evidence has demonstrated the presence of intertissue-communication regulating systemic aging, but the underlying molecular network has not been fully explored. We and others previously showed that two basic helix-loop-helix transcription factors, MML-1 and HLH-30, are required for lifespan extension in several longevity paradigms, including germlineless Caenorhabditis elegans. However, it is unknown what tissues these factors target to promote longevity. Here, using tissue-specific knockdown experiments, we found that MML-1 and its heterodimer partners MXL-2 and HLH-30 act primarily in neurons to extend longevity in germlineless animals. Interestingly, however, the downstream cascades of MML-1 in neurons were distinct from those of HLH-30. Neuronal RNA interference (RNAi)-based transcriptome analysis revealed that the glutamate transporter GLT-5 is a downstream target of MML-1 but not HLH-30. Furthermore, the MML-1-GTL-5 axis in neurons is critical to prevent an age-dependent collapse of proteostasis and increased oxidative stress through autophagy and peroxidase MLT-7, respectively, in long-lived animals. Collectively, our study revealed that systemic aging is regulated by a molecular network involving neuronal MML-1 function in both neural and peripheral tissues.

Conditional Astrocyte Rac1KO Attenuates Hyperreflexia after Spinal Cord Injury.

Spasticity is a hyperexcitability disorder that adversely impacts functional recovery and rehabilitative efforts after spinal cord injury (SCI). The loss of evoked rate-dependent depression (RDD) of the monosynaptic H-reflex is indicative of hyperreflexia, a physiological sign of spasticity. Given the intimate relationship between astrocytes and neurons, that is, the tripartite synapse, we hypothesized that astrocytes might have a significant role in post-injury hyperreflexia and plasticity of neighboring neuronal synaptic dendritic spines. Here, we investigated the effect of selective Rac1KO in astrocytes (i.e., adult male and female mice, transgenic cre-flox system) on SCI-induced spasticity. Three weeks after a mild contusion SCI, control Rac1wt animals displayed a loss of H-reflex RDD, that is, hyperreflexia. In contrast, transgenic animals with astrocytic Rac1KO demonstrated near-normal H-reflex RDD similar to pre-injury levels. Reduced hyperreflexia in astrocytic Rac1KO animals was accompanied by a loss of thin-shaped dendritic spine density on α-motor neurons in the ventral horn. In SCI-Rac1wt animals, as expected, we observed the development of dendritic spine dysgenesis on α-motor neurons associated with spasticity. As compared with WT animals, SCI animals with astrocytic Rac1KO expressed increased levels of the glial-specific glutamate transporter, glutamate transporter-1 in the ventral spinal cord, potentially enhancing glutamate clearance from the synaptic cleft and reducing hyperreflexia in astrocytic Rac1KO animals. Taken together, our findings show for the first time that Rac1 activity in astrocytes can contribute to hyperreflexia underlying spasticity following SCI. These results reveal an opportunity to target cell-specific molecular regulators of H-reflex excitability to manage spasticity after SCI.Significance Statement Spinal cord injury leads to stretch reflex hyperexcitability, which underlies the clinical symptom of spasticity. This study shows for the first time that astrocytic Rac1 contributes to the development of hyperreflexia after SCI. Specifically, astrocytic Rac1KO reduced SCI-related H-reflex hyperexcitability, decreased dendritic spine dysgenesis on α-motor neurons, and elevated the expression of the astrocytic glutamate transporter-1 (GLT-1). Overall, this study supports a distinct role for astrocytic Rac1 signaling within the spinal reflex circuit and the development of SCI-related spasticity.

The high-energy transition state of the glutamate transporter homologue GltPh.

Membrane transporters mediate cellular uptake of nutrients, signaling molecules, and drugs. Their overall mechanisms are often well understood, but the structural features setting their rates are mostly unknown. Earlier single-molecule fluorescence imaging of the archaeal model glutamate transporter homologue GltPh from Pyrococcus horikoshii suggested that the slow conformational transition from the outward- to the inward-facing state, when the bound substrate is translocated from the extracellular to the cytoplasmic side of the membrane, is rate limiting to transport. Here, we provide insight into the structure of the high-energy transition state of GltPh that limits the rate of the substrate translocation process. Using bioinformatics, we identified GltPh gain-of-function mutations in the flexible helical hairpin domain HP2 and applied linear free energy relationship analysis to infer that the transition state structurally resembles the inward-facing conformation. Based on these analyses, we propose an approach to search for allosteric modulators for transporters.

Distinct roles of the Na+ binding sites in the allosteric coupling mechanism of the glutamate transporter homolog, GltPh.

Glutamate transporters carry out the concentrative uptake of glutamate by harnessing the ionic gradients present across cellular membranes. A central step in the transport mechanism is the coupled binding of Na+ and substrate. The sodium coupled Asp transporter, GltPh is an archaeal homolog of glutamate transporters that has been extensively used to probe the transport mechanism. Previous studies have shown that hairpin-2 (HP2) functions as the extracellular gate for the aspartate binding site and plays a key role in the coupled binding of sodium and aspartate to GltPh. The binding sites for three Na+ ions (Na1-3) have been identified in GltPh, but the specific roles of the individual Na+ sites in the binding process have not been elucidated. In this study, we developed assays to probe Na+ binding to the Na1 and Na3 sites and to monitor the conformational switch in the NMDGT motif. We used these assays along with a fluorescence assay to monitor HP2 movement and EPR spectroscopy to show that Na+ binding to the Na3 site is required for the NMDGT conformational switch while Na+ binding to the Na1 site is responsible for the partial opening of HP2. Complete opening of HP2 requires the conformational switch of the NMDGT motif and therefore Na+ binding to both the Na1 and the Na3 sites. Based on our studies, we also propose an alternate pathway for the coupled binding of Na+ and Asp.

Combinatory Actions of Co-transmitters in Dopaminergic Systems Modulate Drosophila Olfactory Memories.

Dopamine neurons (DANs) are extensively studied in the context of associative learning, in both vertebrates and invertebrates. In the acquisition of male and female Drosophila olfactory memory, the PAM cluster of DANs provides the reward signal, and the PPL1 cluster of DANs sends the punishment signal to the Kenyon cells (KCs) of mushroom bodies, the center for memory formation. However, thermo-genetical activation of the PPL1 DANs after memory acquisition impaired aversive memory, and that of the PAM DANs impaired appetitive memory. We demonstrate that the knockdown of glutamate decarboxylase, which catalyzes glutamate conversion to GABA in PAM DANs, potentiated the appetitive memory. In addition, the knockdown of glutamate transporter in PPL1 DANs potentiated aversive memory, suggesting that GABA and glutamate co-transmitters act in an inhibitory manner in olfactory memory formation. We also found that, in γKCs, the Rdl receptor for GABA and the mGluR DmGluRA mediate the inhibition. Although multiple-spaced training is required to form long-term aversive memory, a single cycle of training was sufficient to develop long-term memory when the glutamate transporter was knocked down, in even a single subset of PPL1 DANs. Our results suggest that the mGluR signaling pathway may set a threshold for memory acquisition to allow the organisms' behaviors to adapt to changing physiological conditions and environments.SIGNIFICANCE STATEMENT In the acquisition of olfactory memory in Drosophila, the PAM cluster of dopamine neurons (DANs) mediates the reward signal, while the PPL1 cluster of DANs conveys the punishment signal to the Kenyon cells of the mushroom bodies, which serve as the center for memory formation. We found that GABA co-transmitters in the PAM DANs and glutamate co-transmitters in the PPL1 DANs inhibit olfactory memory formation. Our findings demonstrate that long-term memory acquisition, which typically necessitates multiple-spaced training sessions to establish aversive memory, can be triggered with a single training cycle in cases where the glutamate co-transmission is inhibited, even within a single subset of PPL1 DANs, suggesting that the glutamate co-transmission may modulate the threshold for memory acquisition.

Differential Effects of Astrocyte Manipulations on Learned Motor Behavior and Neuronal Ensembles in the Motor Cortex.

Although motor cortex is crucial for learning precise and reliable movements, whether and how astrocytes contribute to its plasticity and function during motor learning is unknown. Here, we report that astrocyte-specific manipulations in primary motor cortex (M1) during a lever push task alter motor learning and execution, as well as the underlying neuronal population coding. Mice that express decreased levels of the astrocyte glutamate transporter 1 (GLT1) show impaired and variable movement trajectories, whereas mice with increased astrocyte Gq signaling show decreased performance rates, delayed response times, and impaired trajectories. In both groups, which include male and female mice, M1 neurons have altered interneuronal correlations and impaired population representations of task parameters, including response time and movement trajectories. RNA sequencing further supports a role for M1 astrocytes in motor learning and shows changes in astrocytic expression of glutamate transporter genes, GABA transporter genes, and extracellular matrix protein genes in mice that have acquired this learned behavior. Thus, astrocytes coordinate M1 neuronal activity during motor learning, and our results suggest that this contributes to learned movement execution and dexterity through mechanisms that include regulation of neurotransmitter transport and calcium signaling.SIGNIFICANCE STATEMENT We demonstrate for the first time that in the M1 of mice, astrocyte function is critical for coordinating neuronal population activity during motor learning. We demonstrate that knockdown of astrocyte glutamate transporter GLT1 affects specific components of learning, such as smooth trajectory formation. Altering astrocyte calcium signaling by activation of Gq-DREADD upregulates GLT1 and affects other components of learning, such as response rates and reaction times as well as trajectory smoothness. In both manipulations, neuronal activity in motor cortex is dysregulated, but in different ways. Thus, astrocytes have a crucial role in motor learning via their influence on motor cortex neurons, and they do so by mechanisms that include regulation of glutamate transport and calcium signals.

Neuronal small extracellular vesicles carrying miR-181c-5p contribute to the pathogenesis of epilepsy by regulating the protein kinase C-δ/glutamate transporter-1 axis in astrocytes.

Information exchange between neurons and astrocytes mediated by extracellular vesicles (EVs) is known to play a key role in the pathogenesis of central nervous system diseases. A key driver of epilepsy is the dysregulation of intersynaptic excitatory neurotransmitters mediated by astrocytes. Thus, we investigated the potential association between neuronal EV microRNAs (miRNAs) and astrocyte glutamate uptake ability in epilepsy. Here, we showed that astrocytes were able to engulf epileptogenic neuronal EVs, inducing a significant increase in the glutamate concentration in the extracellular fluid of astrocytes, which was linked to a decrease in glutamate transporter-1 (GLT-1) protein expression. Using sequencing and gene ontology (GO) functional analysis, miR-181c-5p was found to be the most significantly upregulated miRNA in epileptogenic neuronal EVs and was linked to glutamate metabolism. Moreover, we found that neuronal EV-derived miR-181c-5p interacted with protein kinase C-delta (PKCδ), downregulated PKCδ and GLT-1 protein expression and increased glutamate concentrations in astrocytes both in vitro and in vivo. Our findings demonstrated that epileptogenic neuronal EVs carrying miR-181c-5p decrease the glutamate uptake ability of astrocytes, thus promoting susceptibility to epilepsy.

Thyroid hormone deficiency affects anxiety-related behaviors and expression of hippocampal glutamate transporters in male congenital hypothyroid rat offspring.

Thyroid hormones are crucial for brain development and their deficiency during fetal and postnatal periods can lead to mood and cognitive disorders. We aimed to examine the consequences of thyroid hormone deficiency on anxiety-related behaviors and protein expression of hippocampal glutamate transporters in congenital hypothyroid male offspring rats. Possible beneficial effects of treadmill exercise have also been examined. Congenital hypothyroidism was induced by adding propylthiouracil (PTU) to drinking water of pregnant Wistar rats from gestational day 6 until the end of the weaning period (postnatal day 28). Next, following 4 weeks of treadmill exercise (5 days per week), anxiety-related behaviors were examined using elevated plus maze (EPM) and light/dark box tests. Thereafter, protein expression of astrocytic (GLAST and GLT-1) and neuronal (EAAC1) glutamate transporters were measured in the hippocampus by immunoblotting. Hypothyroid rats showed decreased anxiety-like behavior, as measured by longer time spent in the open arms of the EPM and in the light area of the light/dark box, compared to control rats. Hypothyroid rats had significantly higher GLAST and GLT-1 and lower EAAC1 protein levels in the hippocampus than did the euthyroid rats. Following exercise, anxiety levels decreased in the euthyroid group while protein expression of EAAC1 increased and returned to normal levels in the hypothyroid group. Our findings indicate that thyroid hormone deficiency was associated with alterations in protein expression of glutamate transporters in the hippocampus. Up-regulation of hippocampal GLAST and GLT-1 could be at least one of the mechanisms associated with the anxiolytic effects of congenital hypothyroidism.

Linking function to global and local dynamics in an elevator-type transporter.

Transporters cycle through large structural changes to translocate molecules across biological membranes. The temporal relationships between these changes and function, and the molecular properties setting their rates, determine transport efficiency-yet remain mostly unknown. Using single-molecule fluorescence microscopy, we compare the timing of conformational transitions and substrate uptake in the elevator-type transporter GltPh We show that the elevator-like movements of the substrate-loaded transport domain across membranes and substrate release are kinetically heterogeneous, with rates varying by orders of magnitude between individual molecules. Mutations increasing the frequency of elevator transitions and reducing substrate affinity diminish transport rate heterogeneities and boost transport efficiency. Hydrogen deuterium exchange coupled to mass spectrometry reveals destabilization of secondary structure around the substrate-binding site, suggesting that increased local dynamics leads to faster rates of global conformational changes and confers gain-of-function properties that set transport rates.

Elevation of Extracellular Glutamate by Blockade of Astrocyte Glutamate Transporters Inhibits Cocaine Reinforcement in Rats via a NMDA-GluN2B Receptor Mechanism.

It is well established that glutamate plays an important role in drug-induced and cue-induced reinstatement of drug seeking. However, the role of glutamate in drug reward is unclear. In this study, we systemically evaluated the effects of multiple glutamate transporter (GLT) inhibitors on extracellular glutamate and dopamine (DA) in the nucleus accumbens (NAc), intravenous cocaine self-administration, intracranial brain-stimulation reward (BSR), and reinstatement of cocaine seeking in male and female rats. Among the five GLT inhibitors we tested, TFB-TBOA was the most potent. Microinjections of TFB-TBOA into the NAc, but not the ventral tegmental area (VTA), or dorsal striatum (DS), dose-dependently inhibited cocaine self-administration under fixed-ratio and progressive-ratio (PR) reinforcement schedules, shifted the cocaine dose-response curve downward, and inhibited intracranial BSR. Selective downregulation of astrocytic GLT-1 expression in the NAc by GLT-1 antisense oligonucleotides also inhibited cocaine self-administration. The reduction in cocaine self-administration following TFB-TBOA administration was NMDA GluN2B receptor dependent, and rats self-administering cocaine showed upregulation of GluN2B expression in NAc DA- and cAMP-regulated phosphoprotein 32 (DARPP-32)-positive medium-spiny neurons (MSNs). In contrast, TFB-TBOA, when locally administered into the NAc, VTA, or ventral pallidum (VP), dose-dependently reinstated cocaine-seeking behavior. Intra-NAc TFB-TBOA-evoked drug-seeking was long-lasting and NMDA/AMPA receptor dependent. These findings, for the first time, indicate that glutamate in the NAc negatively regulates cocaine's rewarding effects, while an excess of glutamate in multiple brain regions can trigger reinstatement of drug-seeking behavior.SIGNIFICANCE STATEMENT It is well known that glutamate plays an important role in relapse to drug seeking. However, the role of glutamate in drug reward is less clear. Here, we report that TFB-TBOA, a highly potent glutamate transporter (GLT) inhibitor, dose-dependently elevates extracellular glutamate and inhibits cocaine self-administration and brain-stimulation reward (BSR), when administered locally into the nucleus accumbens (NAc), but not other brain regions. Mechanistic assays indicate that cocaine self-administration upregulates NMDA-GluN2B receptor subtype expression in striatal dopaminoceptive neurons and activation of GluN2B by TFB-TBOA-enhanced glutamate inhibits cocaine self-administration. TFB-TBOA also reinstates cocaine-seeking behavior when administered into the NAc, ventral tegmental area (VTA), and ventral pallidum (VP). These findings demonstrate that glutamate differentially regulates cocaine reward versus relapse, reducing cocaine reward, while potentiating relapse to cocaine seeking.

EphA4/ephrinA3 reverse signaling mediated downregulation of glutamate transporter GLAST in Müller cells in an experimental glaucoma model.

Deficiency of glutamate transporter GLAST in Müller cells may be culpable for excessive extracellular glutamate, which involves in retinal ganglion cell (RGC) damage in glaucoma. We elucidated how GLAST was regulated in rat chronic ocular hypertension (COH) model. Western blot and whole-cell patch-clamp recordings showed that GLAST proteins and GLAST-mediated current densities in Müller cells were downregulated at the early stages of COH. In normal rats, intravitreal injection of the ephrinA3 activator EphA4-Fc mimicked the changes of GLAST in COH retinas. In purified cultured Müller cells, EphA4-Fc treatment reduced GLAST expression at mRNA and protein levels, which was reversed by the tyrosine kinase inhibitor PP2 or transfection with ephrinA3-siRNA (Si-EFNA3), suggesting that EphA4/ephrinA3 reverse signaling mediated GLAST downregulation. EphA4/ephrinA3 reverse signaling-induced GLAST downregulation was mediated by inhibiting PI3K/Akt/NF-κB pathways since EphA4-Fc treatment of cultured Müller cells reduced the levels of p-Akt/Akt and NF-κB p65, which were reversed by transfecting Si-EFNA3. In Müller cells with ephrinA3 knockdown, the PI3K inhibitor LY294002 still decreased the protein levels of NF-κB p65 in the presence of EphA4-Fc, and the mRNA levels of GLAST were reduced by LY294002 and the NF-κB inhibitor SN50, respectively. Pre-injection of the PI3K/Akt pathway activator 740 Y-P reversed the GLAST downregulation in COH retinas. Western blot and TUNEL staining showed that transfecting of Si-EFNA3 reduced Müller cell gliosis and RGC apoptosis in COH retinas. Our results suggest that activated EphA4/ephrinA3 reverse signaling induces GLAST downregulation in Müller cells via inhibiting PI3K/Akt/NF-κB pathways, thus contributing to RGC damage in glaucoma.

Milestone Review: Excitatory amino acid transporters - Beyond their expected function.

Glutamate is the major excitatory neurotransmitter in the vertebrate brain, it is critically involved in the function and dysfunction of the central nervous system. The molecular cloning of its ionotropic receptors in the last decade of the past century increased exponentially the interest in this neurotransmitter system. Since then, a plethora of knowledge of the structure, function, and regulation of its receptors and transporters has advanced our understanding of glutamate-mediated neurochemical transactions. Moreover, the characterization of glial glutamate receptors together with the compulsory participation of surrounding astrocytes in glutamate turnover and in the known metabolic coupling with neurons has supported what is now known as the tripartite synapses. The molecular characterization of the various glutamate transporters has also been fundamental for the involvement of glial cells in glutamatergic synapses. Using radial glial cultures, over the years, we have demonstrated an alternative glutamate-mediated signaling system triggered by sodium-dependent glutamate transporters. A detailed account of these findings and the signaling through other glutamate transporters are presented here. The role of this signaling system in the context of glutamatergic transmission is discussed as well as the future directions in the field.

Epigenome-wide association study of alcohol consumption in N = 8161 individuals and relevance to alcohol use disorder pathophysiology: identification of the cystine/glutamate transporter SLC7A11 as a top target.

Alcohol misuse is common in many societies worldwide and is associated with extensive morbidity and mortality, often leading to alcohol use disorders (AUD) and alcohol-related end-organ damage. The underlying mechanisms contributing to the development of AUD are largely unknown; however, growing evidence suggests that alcohol consumption is strongly associated with alterations in DNA methylation. Identification of alcohol-associated methylomic variation might provide novel insights into pathophysiology and novel treatment targets for AUD. Here we performed the largest single-cohort epigenome-wide association study (EWAS) of alcohol consumption to date (N = 8161) and cross-validated findings in AUD populations with relevant endophenotypes, as well as alcohol-related animal models. Results showed 2504 CpGs significantly associated with alcohol consumption (Bonferroni p value < 6.8 × 10-8) with the five leading probes located in SLC7A11 (p = 7.75 × 10-108), JDP2 (p = 1.44 × 10-56), GAS5 (p = 2.71 × 10-47), TRA2B (p = 3.54 × 10-42), and SLC43A1 (p = 1.18 × 10-40). Genes annotated to associated CpG sites are implicated in liver and brain function, the cellular response to alcohol and alcohol-associated diseases, including hypertension and Alzheimer's disease. Two-sample Mendelian randomization confirmed the causal relationship of consumption on AUD risk (inverse variance weighted (IVW) p = 5.37 × 10-09). A methylation-based predictor of alcohol consumption was able to discriminate AUD cases in two independent cohorts (p = 6.32 × 10-38 and p = 5.41 × 10-14). The top EWAS probe cg06690548, located in the cystine/glutamate transporter SLC7A11, was replicated in an independent cohort of AUD and control participants (N = 615) and showed strong hypomethylation in AUD (p < 10-17). Decreased CpG methylation at this probe was consistently associated with clinical measures including increased heavy drinking days (p < 10-4), increased liver function enzymes (GGT (p = 1.03 × 10-21), ALT (p = 1.29 × 10-6), and AST (p = 1.97 × 10-8)) in individuals with AUD. Postmortem brain analyses documented increased SLC7A11 expression in the frontal cortex of individuals with AUD and animal models showed marked increased expression in liver, suggesting a mechanism by which alcohol leads to hypomethylation-induced overexpression of SLC7A11. Taken together, our EWAS discovery sample and subsequent validation of the top probe in AUD suggest a strong role of abnormal glutamate signaling mediated by methylomic variation in SLC7A11. Our data are intriguing given the prominent role of glutamate signaling in brain and liver and might provide an important target for therapeutic intervention.

Upregulation of glutamate transporter 1 by mTOR/Akt pathway in astrocyte culture during oxygen-glucose deprivation and reoxygenation.

Astrocyte-specific glutamate transporter subtype 1 (GLT-1) plays an important role in influencing glutamate excitatory toxicity and preventing the death of excitatory toxic neurons. Although the mammalian target of rapamycin (mTOR)/protein kinase B(Akt)/nuclear factor kappa B signaling cascade is involved in the upregulation of astrocytic GLT-1 in oxygen-glucose deprivation (OGD), it is unclear whether the mTOR/Akt pathway is involved in astrocytic GLT-1 upregulation in OGD and reoxygenation (OGD/R). In this study, we found that the treatment of cultured astrocytes with rapamycin and triciribine led to the decreased astrocytes' protrusions, smaller nuclei, and an increased apoptotic rate. The inhibitors of mTOR complex 1 significantly increased the expression levels of phosphorylated Akt-Ser473 (p-Akt), phosphorylated Akt-Thr308(p-Akt), and GLT-1, while Akt-specific inhibitors blocked GLT-1 expression, suggesting that the mTOR/Akt pathway is involved in GLT-1 upregulation. We further demonstrated that astrocytes under OGD/R adapted to environmental changes through the mTOR/Akt pathway, mainly by altering cell morphology and apoptosis and upregulating the expression levels of p-Akt and GLT-1. Our results suggested that astrocytes may adapt to short-term ischemic-reperfusion injury by regulating cell morphology, apoptosis and GLT-1 upregulation.

Marrow Mesenchymal Stem Cell-Derived Exosomes Upregulate Astrocytic Glutamate Transporter-1 Expression via miR-124/mTOR Pathway against Oxygen-Glucose Deprivation/Reperfusion Injury.

BACKGROUND: Experimental investigations have reported the efficacy of marrow mesenchymal stem cell-derived exosomes (MSC-Exos) for the treatment of ischemic stroke. The therapeutic mechanism, however, is still unknown. The purpose of the study is to show whether MSC-Exos increases astrocytic glutamate transporter-1 (GLT-1) expression in response to ischemic stroke and to investigate further mechanisms. METHODS AND RESULTS: An in vitro ischemia model (oxygen-glucose deprivation/reperfusion, OGD/R) was used. MSC-Exos was identified by Western blot (WB) and transmission electron microscopy (TEM). To further investigate the mechanism, MSC-Exos, miR-124 inhibitor, and mimics, and a mTOR pathway inhibitor (rapamycin, Rap) were used. The interaction between GLT-1 and miR-124 was analyzed by luciferase reporter assay. The GLT-1 RNA expression and miR-124 was assessed by quantitative real-time polymerase chain reaction (qRTPCR). The protein expressions of GLT-1, S6, and pS6 were detected by WB. Results demonstrated that MSC-Exos successfully inhibited the decrease of GLT-1 and miR-124 expression and the increase of pS6 expression in astrocytes after OGD/R. miR-124 inhibitor suppressed the effect of MSC-Exos on GLT-1 upregulation after OGD/R. Rapamycin notably decreased pS6 expression with significantly higher GLT-1 expression in astrocytes injured by OGD/R. Luciferase activity of the reporter harboring the wild-type or mutant GLT-1 3'UTR was not inhibited by miR-124 mimics. Further results showed that the inhibiting effect of MSC-Exos on pS6 expression and promoting effect of MSC-Exos on GLT-1 expression could be reversed by miR-124 inhibitor after OGD/R; meanwhile, the above conditions could be reversed again by rapamycin. CONCLUSIONS: Results show that miR-124 and the mTOR pathway are involved in regulation of MSC-Exos on GLT-1 expression in astrocytes injured by OGD/R. miR-124 does not directly target GLT-1. MSC-Exos upregulates GLT-1 expression via the miR-124/mTOR pathway in astrocytes injured by OGD/R.

ß_lactam antibiotics against drug addiction: A novel therapeutic option.

Drug addiction as a problem for the health of the individual and the society is the result of a complex process in which there is an interaction between brain nuclei and neurotransmitters (such as glutamate). ß-lactam antibiotics, due to their enhancing properties on the glutamate transporter glutamate transporter-1, can affect and counteract the addictive mechanisms of drugs through the regulation of extracellular glutamate. Since glutamate is a key neurotransmitter in the development of drug addiction, it seems that ß-lactams can be considered as a promising treatment for addiction. However, more research in this field is necessary to identify other mechanisms involved in their effectiveness. This article is a review of the studies conducted on the effect of ß-lactam administration in preventing the development of drug addiction, as well as their possible cellular and molecular mechanisms. This review suggests the clinical use of ß-lactam antibiotics that have weak antimicrobial properties (such as clavulanic acid) in the treatment of drug dependence.

Neuroprotective Potential of L-Glutamate Transporters in Human Induced Pluripotent Stem Cell-Derived Neural Cells against Excitotoxicity.

Human induced pluripotent stem cell (hiPSC)-derived neural cells have started to be used in safety/toxicity tests at the preclinical stage of drug development. As previously reported, hiPSC-derived neurons exhibit greater tolerance to excitotoxicity than those of primary cultures of rodent neurons; however, the underlying mechanisms remain unknown. We here investigated the functions of L-glutamate (L-Glu) transporters, the most important machinery to maintain low extracellular L-Glu concentrations, in hiPSC-derived neural cells. We also clarified the contribution of respective L-Glu transporter subtypes. At 63 days in vitro (DIV), we detected neuronal circuit functions in hiPSC-derived neural cells by a microelectrode array system (MEA). At 63 DIV, exposure to 100 µM L-Glu for 24 h did not affect the viability of neural cells. 100 µM L-Glu in the medium decreased to almost 0 µM in 60 min. Pharmacological inhibition of excitatory amino acid transporter 1 (EAAT1) and EAAT2 suppressed almost 100% of L-Glu decrease. In the presence of this inhibitor, 100 µM L-Glu dramatically decreased cell viability. These results suggest that in hiPSC-derived neural cells, EAAT1 and EAAT2 are the predominant L-Glu transporters, and their uptake potentials are the reasons for the tolerance of hiPSC-derived neurons to excitotoxicity.